The Role Of Autophagy In The Effects Of Anti-keloid Activity By MTOR Inhibitor

Objective:The mTOR signal is closely related to cell proliferation,growth,metabolism and other activities.More importantly,mTOR is an important regulator on autophagy which is also closely related to the biological functions of cell proliferation.However,the role of the mTOR/autophagy signaling pathway in keloid remains unclear One of purposes of the study was to investigate the mTOR activity and autophagy level in keloid patients.Then the second purpose was to explore the biological effects of mTOR inhibitors rapamycin and KU-0063794 in keloid-derived fibroblasts.Finally,we performed further exploration on the role of autophagy in the functional effects of mTOR inhibitors.Methods:1.The expression of mTOR,downstream signal molecule and autophagy-related proteins LC3,ULK1,ATG7,ATG9A and ATG12 were detected by immunohistochemical staining and Western blot assay in keloid tissues and isolated fibroblasts respectively.Finally,the fibroblasts were stained with acridine orange(AO)to observe the formation of acidic vesicles in the cytoplasm.2.Keloid-derived fibroblasts were treated with mTOR inhibitor,including 100 nM rapamycin or 5 ?M KU-0063794 at different time points.The effects on cell proliferation were detected by CCK-8 and BrdU incorporation experiments respectively.Annexin V-EGFP/PI double staining was conducted to detect cell apoptosis by flow cytometry.Western blot method was used to detect the cleavage of caspase 3 and PARP.In vitro two-dimensional migration assay was performed to examine its effect on cell migration.The synthesis of collagen type ? and type ? were detected by Western blot assay.3.To determine the effect of rapamycin and KU-0063794 on autophagic flow in keloid-derived fibroblasts,Western blot assay was used to detect the expression of LC3.Keloid-derived fibroblasts were pretreated with wortmannin,an autophagy inhibitor,or transfected with si-ATG5 to block the autophagic flow.Then,rapamycin and KU-0063794 were administered to treat different time points.The effects on cell proliferation,migration and collagen synthesis were detected again by the same methods.Results:1.Results showed that increased expression of mTOR,S6,p-mTOR Ser2448,p-S6 Ser235/236 and decreased expression of autophagy marker protein LC3 were found in keloid tissues compared with the control group by immunohistochemical staining and Western blot assay.In vitro experiments,it showed that the expression of p-S6 Ser235/236 and p-ULK1 Ser757 was increased,while LC3 was decreased in keloid-derived fibroblasts compared with normal fibroblasts.The fluorescence staining of acridine orange showed that the number of acidic vesicles in keloid-derived fibroblasts were decreased.2.Rapamycin and KU-0063794 could inhibit the proliferation,migration and collagen synthesis in keloid-derived fibroblasts,but had no effect on apoptosis.In addition,the inhibitory effect of dual mTORC1/2 inhibitor KU-0063794 was stronger than that of mTORCl inhibitor rapamycin.3.Rapamycin and KU-0063794 could induce autophagic responses and pretreatment with wortmannin or transfection with si-ATG5 could block the autophagic flow induced by rapamycin and KU-0063794 in keloid-derived fibroblasts.However,the inhibitory effects on proliferation,migration and collagen synthesis induced by rapamycin and KU-0063794 could not be altered by blocking autophagic flow.Conclusions:The abnormal activation of mTOR signaling pathway was involved in the pathogenesis of keloid accompanied by the inhibition of autophagy.The mTOR inhibitors including rapamycin and KU-0063794 had significant biological effects on the proliferation,migration and collagen synthesis in keloid-derived fibroblasts which showed potential therapeutic value.Our study found that in addition to inhibiting proliferation,migration and collagen synthesis,mTOR inhibitors can also induce autophagy of keloid-derived fibroblasts.But,autophagy,a key downstream signal of mTOR,does not mediate the above effects of mTOR inhibitors.